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Free Energy Landscape of Lysozyme: Multiple Near-Native Conformational States and Rollover in the Urea Dependence of Folding Energy
journal contribution
posted on 2014-06-19, 00:00 authored by U. Mahammad Yasin, Pulikallu Sashi, Abani K. BhuyanDeviation
from linearity of the equilibrium folding free energy
(ΔG) of proteins along the reaction coordinate
is scarcely known. Optical spectroscopic observables and NMR-measured
average molecular dimensional property of lysozyme with urea at pH
5 reveal that ΔG rolls over from linearity
under mild to strongly native-like conditions. The urea dependence
of ΔG is graphed in the 0–7 M range
of the denaturant by employing a series of guanidine hydrochloride
(GdnHCl)-induced equilibrium unfolding transitions, each in the presence
of a fixed level of urea. The observed linear dependence of ΔG on urea under denaturing conditions begins to deviate
as moderately native-like conditions are approached and eventually
rolls over under strongly native-like conditions. This is atypical
of the upward curvature in the ΔG vs denaturant
plot predicted by the denaturant binding model. On increasing the
denaturant concentration from 0 to 5 M, the hydrodynamic radius of
lysozyme shrinks by ∼2 Å. We suggest subdenaturing levels
of urea affect the population distribution among multiple near-native
isoenergetic conformational states so as to promote them sequentially
with increments of the denaturant. We use a multiple-state sequential
model to show that the keel over of ΔG occurs
due to these near-native alternative states in the native ensemble
used for defining the unfolding equilibrium constant (KU), which we assume to vary linearly with urea. The results
and the model appear to indicate a rugged flat bottom in the free
energy landscape wherein population distribution of native-like states
is modulated by urea-affected interstate motions.